ABSTRACT Melanoma is the most lethal form of skin cancer. If discovered early, it is usually cured with surgery; however, treatment options are limited for metastatic melanoma, with a 5-year survival of only 15-20%. Although immunotherapy as a first-line treatment option has improved survival, individual response is uneven, possibly due in part to the quality of protective tumor infiltrating lymphocytes (TILs) impacted by treatment intervention. There is critical need to: 1) identify patients with an aggressive phenotype; 2) improve therapies to increase overall survival of such patients; and 3) develop improved monitoring means to discern whether patients are responding to therapy. Here we propose a multi-faceted approach for imaging and therapy of metastatic melanoma, to be tested in mouse models of primary and metastatic disease. There is great interest in targeting very late antigen-4 (VLA-4; also called integrin ?4?1) for cancer imaging and therapy for multiple myeloma and melanoma, where it plays a facilitating role in tumor growth, angiogenesis and metastasis by promoting adhesion and migration of cancer cells. In humans, increased expression of VLA-4 in melanoma correlates with development of metastasis. We previously showed that high-affinity peptidomimetic VLA-4 targeted agents labeled with 68Ga (for Positron Emission Tomography (PET); T1/2 = 68 min) and 177Lu (for radionuclide therapy; T1/2 = 6.7 d) are avidly taken up by B16F10 melanoma tumors in mice. A recently published study showed dramatic efficacy from combining external beam irradiation (XRT) with the combination immunotherapy agents targeting CTLA-4 and PD1/PD-L1 therapy in B16F10 tumor-bearing mice. As XRT is not optimal for treating widely disseminated or micrometastatic disease, we aim to improve upon this important finding by investigating VLA-4 targeted, systemic, radiotherapy with 177Lu-DOTA-PEG4-LLP2A (177Lu-LLP2A), in combination with anti-CTLA-4 and anti-PD-1 immunotherapy. We will also develop PET imaging tracers targeting PD-L1 and CD8 to provide real-time, non-invasive monitoring of tumor cells, myeloid- derived cells and T cells in response to therapy. We hypothesize that targeted radiotherapy with 177Lu-LLP2A, combined with dual anti-CTLA-4 and anti-PD-1 immunotherapy, will be highly effective in treating melanoma tumor-bearing mice, and that imaging of PD-1 and CD8+ T-cells will allow the monitoring of early ?clinical? responses to therapy. To address our hypotheses, we propose the following aims: 1) validate uptake of 68Ga- and 177Lu-labeled LLP2A in a mouse model of melanoma (BP20) where the tumors have the common BRAFV600E mutation, and in human tumors derived from patient melanoma metastases; 2) determine the optimal treatment strategy comparing 177Lu-LLP2A and XRT in combination with dual immunotherapy in B16F10 subcutaneous and disseminated tumors, and in BP20 tumors with the BRAFV600E mutation; and 3) develop and validate anti-mouse PD-L1 minibodies, and anti-mouse CD8 single domain antibody (sdAb) PET imaging agents labeled with 64Cu (T1/2 = 12.7 h) and 68Ga (T1/2 = 68 min), respectively. The combination therapy and PET imaging of early response will be performed. If successful, we will demonstrate that combining targeted radionuclide therapy and immunotherapy can effectively treat advanced-stage melanoma, while simultaneously identifying a panel of PET tracers for non-invasive monitoring of treatment efficacy.